Our studies have addressed several major questions: 1) We have demonstrated that human Foxp3+ T cells, activated with plate-bound anti-hCD3, are almost as potent inhibitors of the proliferation of mouse CD4+Foxp3- responders (stimulated with soluble anti-mouse CD3 and mouse DC), as mouse CD4+Foxp3+ Treg. This result strongly suggests that at least in vitro a major component of the suppressive function of Foxp3+ Treg is preserved across the species. Human Treg can also inhibit the upregulation of costimulatory molecules on mouse DCs. One advantage of this model is that it allows us to attempt to block the suppressive capacity of the human T cells with mAbs to human cell surface antigens that will not interfere with the activation of the mouse responder cells. We have generated large panels of mAbs to activated human Treg and are in the process of testing these antibodies for their capacity to reverse the inhibitory effects of the human Treg. The antibodies are being are also being tested for their ability to stain different human T cell populations and are being used to characterize the biochemical properties of their target antigen. 2) Although functional populations of Foxp3+ Treg can readily be induced by stimulation of mouse T cells via the T cell receptor in the presence of TGF-beta, we have thus far been unable to obtain functional Treg cells using this protocol even though the cells express relatively high levels of Foxp3. We are exploring different conditions for the induction of functional Foxp3+ Treg cells by stimulating different populations of naive human T cells under different conditions of co-stimulation. 3.We have recently described that both mouse and human Foxp3+ T cell populations can be divided into two distinct subsets based on expression of the transcription factor, Helios. The majority (70%) of Foxp3+ cells co-express Helios and are likely to be thymic derived nTregs, while 30% are Helios- and appear to be peripherally induced iTregs. We have compared the TCR Vbeta repertoire in Foxp3- vs. Foxp3+ CD4+ T cells and also compared the repertoire of the Helios+Foxp3+ and the Helios-Foxp3+ subsets. PBMCs from normal donors were obtained from the DTM at the NIH. PBMCs from cGHVD patients (on average 3 years post HSCT) were obtained from a cohort followed at the NCI. The IOTest Vbeta Kit from Beckman Coulter was used to assess usage of 24 different Vbetas by FACS in combination with co-staining for CD4, FoxP3, and Helios. Overall TCR Vbeta repertoires for FoxP3- vs. FoxP3+ CD4 T cells were similar both in cGVHD patients and in young and older healthy donors. This result is similar to previous published observations. In a number of cGVHD patients and older individuals, there was a striking difference in TCR repertoire usage between the Helios+Foxp3+ and Helios-Foxp3+ subpopulations consistent with the possibility that the Helios-Foxp3+ cells were peripherally induced in response to an environmental or an infectious exposure. Studies are currently under way to identify additional markers that uniquely identify Helios-FoxP3+ cells to facilitate their isolation, functional analysis, and antigen specificity.